Targeting activated integrin alphavbeta3 with patient-derived antibodies impacts late-stage multiorgan metastasis

Abstract

Advanced metastatic disease is difficult to manage and specific therapeutic targets are rare. We showed earlier that metastatic breast cancer cells use the activated conformer of adhesion receptor integrin alphavbeta3 for dissemination. We now investigated if targeting this form of the receptor can impact advanced metastatic disease, and we analyzed the mechanisms involved. Treatment of advanced multi-organ metastasis in SCID mice with patient-derived scFv antibodies specific for activated integrin alphavbeta3 caused stagnation and regression of metastatic growth. The antibodies specifically localized to tumor lesions in vivo and inhibited alphavbeta3 ligand binding at nanomolar levels in vitro. At the cellular level, the scFs associated rapidly with high affinity alphavbeta3 and dissociated extremely slowly. Thus, the scFvs occupy the receptor on metastatic tumor cells for prolonged periods of time, allowing for inhibition of established cell interaction with natural alphavbeta3 ligands. Potential apoptosis inducing effects of the antibodies through interaction with caspase-3 were studied as potential additional mechanism of treatment response. However, in contrast to a previous concept, neither the RGD-containing ligand mimetic scFvs nor RGD peptides bound or activated caspase-3 at the cellular or molecular level. This indicates that the treatment effects seen in the animal model are primarily due to antibody interference with alphavbeta3 ligation. Inhibition of advanced metastatic disease by treatment with cancer patient derived single chain antibodies against the activated conformer of integrin alphavbeta3 identifies this form of the receptor as a suitable target for therapy.

Figure 1. Antibody binding validation and routes of administration

(A) Before use in animals, the binding properties of each scFv phage batch were analyzed by flow cytometry on tumor cells expressing high affinity integrin αvβ3. scFv phage was tested in the presence of calcium on M21 human melanoma cells that carry activated αvβ3 and on MDA-MB 435 cells which express mutant αvβ3_D723R. (B) scFv phage organ distribution in the mouse model. Phage were injected i.v., i.p. or applied intranasally to non-tumor bearing mice to determine phage organ distribution 24 h later.

Figure 2. Lung metastases regress in response to treatment with scFv 1 or scFv 5

F-luc tagged MDA-MB-435 cells were injected i.v. and lesion development monitored by non-invasive bioluminescence imaging (photons/second/cm²) over time. Treatment with scFv phage (5×10¹⁰ per dose) started on day 56 post tumor cell injection. (A) Examples of non- invasive bioluminescence imaging of representative animals that had received 1×10⁵ tumor cells before treatment on day 49, at treatment onset on day 56, and after 4 doses of treatment on day 63. Reduced progression in lung lesions is seen after treatment with scFv1 but not with Wt-phage. (B) Response to treatment given every 48 h (4 doses). Fold-changes of lesion growth were calculated based on growth during 7 days before treatment compared to 7 days under treatment. ScFv1-phage treatment yielded a 57% animal response rate in lung burden and one animal with stabilization of lesion growth. ScFv5-phage treatment resulted in a 60% animal response rate for lung burden. Wt-phage gave no reduction in tumor growth. (C) Non-invasive bioluminescence imaging of lung burden (photons/second/cm²) over time before and during treatment. Animals were treated every 48h for (4 doses). Lung lesion growth was monitored pre-treatment (day 49-56) and during treatment until day 63 (the end of treatment). Dashed vertical lines indicate the start of treatment on day 56. Animals responding to treatment are colored. (D) Response of mice with very advanced metastatic burden, induced by injecting 2.5×10⁵ tumor cells, to treatment given every 24h (8 doses). Treatment with Wt-phage gave no response, whereas regression and reduced tumor progression was seen in the scFv treated animals. ScFv1-phage treatment yielded a response in 75% of the animals, and scFv5-phage in 25%. Animals responding to treatment are colored.

Figure 3. Examples of extrapulmonary lesion regression under treatment with scFv 1

(A) F-luc tagged, in vivo selected and highly metastatic MDA-MB-435-met cells were injected i.v. to induce multiorgan metastasis. Metastatic progression was monitored by non-invasive bioluminescence imaging (photons/second/cm²) over time. In these examples, the location of renal lesions is circled and shown 3 days before treatment, at the beginning of treatment, and after 3 daily doses of scFv1 phage. (B) Fold-change in renal lesion growth, calculated based on signal change during 3 days before and 3 days under treatment.

Figure 4. Localization of scFv1 or scFv 5 phage to areas in and around metastatic lesions in mice with multi-organ metastasis

(A) Immunohistochemical detection of phage homing to metastases in the lungs and lymph node. ScFv phage, or wt control phage, were injected i.p. daily for 7 days into tumor bearing mice. 24h after the last scFv dose, the animals were terminally perfused and frozen tissue sections stained with mAb 29.7 (dark blue), specific for human CD44 indicating the tumor cells, and anti-M13 mAb to detect phage (DAB, brown). Animals treated with wt-phage did not show phage localization to tumor metastases (left panels), but lung metastasis from scFv5-phage treated animals showed phage localization in the tumor proximity as well as within the lung lesion (middle). A lymph node metastasis from an animal treated with scFv1-phage showed phage localizing to the tumor bulk as well as to the outer border of the lesion (right). Controls treated with secondary antibody and substrate did not show specific staining in or around any lesions. Scale bars indicate 100μm. (B) Fluorescence microscopy detecting phage (red). (Upper row) M13 phage was detected in and around lung metastases of scFv1-phage treated animals (right), as well as within the near proximity of the tumor lesions (second right), with minimal phage signal in the unaffected lung tissue away from a tumor lesion (left). Lymph node metastasis showed scFv1-phage localization in the tumor mass and within the tumor proximity (second left, top and bottom). Animals treated with wt-phage showed no specific staining in tumor lesions. Only a weak signal was sometimes seen in non-tumor bearing parts of the tissues, comparable to that seen for scFv phage in unaffected areas of lung tissue (lower left). Using optical sectioning and deconvolution of z-stack images, scFv phage was specifically detected associated with tumor cells (lower second right). Scale bars indicate 100 μm.

Figure 5. Effects of scFv treatment on tumor cells in vivo and host cell infiltration

(A) Ki67 staining of lung metastases treated with wt-phage or scFv1/5-phage (top panel). Lesions in animals responding to treatment showed fewer proliferating cells. Percent area covered by Ki67 signal relative to lesion area measured. H&E staining of the lesions (lower panel). (B) F4/80 macrophage staining of lung metastases in mice treated with wt-phage or scFv1/5-phage. An increased infiltrate was seen in lesions responding to treatment. Percent area covered by F4/80 signal relative to lesion area measured. Scale bars indicate 100 μm in all sections.

Figure 6. Inhibitory and binding properties of scFv 1 and scFv5

(A) Biotinylated natural ligands of αvβ3: vitronectin (VN), fibronectin (FN) or fibrinogen (Fg) (10μg/ml) were incubated with purified immobilized αvβ3 receptor protein in TBS containing Ca²⁺, Mg²⁺, and Mn²⁺ (1mM each), in the presence of increasing concentrations of purified scFv protein. A non-function blocking scFv directed against the integrin v subunit, scFv 20, was used as a control. (B) Flow cytometric binding analysis of fluorescinated scFv5 protein and MDA-MB 435 human tumor cells, expressing either activated high-affinity αvβ3_D723R or non-activated αvβ3_WT. Binding was experimentally maximized in the presence of Mn²⁺, known to induce a high affinity state in integrin heterodimers. Using a Mn²⁺ concentration (25 μM) that supports suboptimal scFv5 binding to tumor cells expressing activated αvβ3_D723R, antibody was titrated to determine binding saturation. (C) Kinetics of scFv cell association and dissociation were analyzed by flow cytometry with FITC-labeled scFv protein at half maximal Mn²⁺ concentration and saturating scFv concentration, using MDA-MB-435_β3D723R cells. (Left) Association time was measured after removing unbound ligand in 10 min intervals. (Right) Dissociation was determined after allowing cells to bind scFv for 1 h to reach binding saturation, followed by removal of unbound ligand, washing and measuring scFv that remained bound in 10 min intervals. FITC-labeled fibrinogen (Fg) was used as a natural ligand for comparison. All incubations were done on ice using ice-cold buffers to prevent scFv or ligand internalization. No binding was detected in the absence of divalent cations.

Figure 7. Caspase-3 expression in the target tumor cells and analysis of caspase-3 binding and activation by RGD-containing scFv1 and scFv5

(A) (Top) Western blot analysis of caspase-3 expression in MDA-MB-435, MCF-7 (negative control) and SKBR-3 (positive control) cells. (Bottom) Verification for procaspase-3 by immunoprecipitation of caspase-3 from tumor cell lysates. (B) ELISA-based analysis of caspase-3 and scFv antibody interaction. Plates were coated with recombinant pro-caspase-3 or BSA (negative control) or scFv 5 (positive control). Addition of RGD-containing scFv 5 or RGE-containing scFv Mut 5 antibody, at concentrations as indicated showed no specific binding to caspase-3. (C) Analysis of caspase-3 activation by scFv antibodies. Hypotonic cell lysates, depleted of mitochondria, were combined with either scFv 5 or scFvMut 5 (4 μM), or RGD or RAD peptides (1 mM) (left panel). Cytochrome c/dATP were used as positive control and PBS as negative control in a bioluminescence assay. Cytochrome c and dATP were able to activate caspase-3 in the lysates, whereas all other samples showed only background signal. A higher scFv concentration was used to verify the lack of activation (10 μM) in the presence of Mg²⁺ (right panel). (D) Continuous measurement of caspase-3 activity in cell lysates based on chromogenic substrate reaction. Cleavage of colorimetric capsase 3 substrate N-acetyl-Asp-Glu-Val-Asp-p-nitroanilide (Ac-DEVD-pNA) was measured after combining hypotonic cell lysates with either scFv 5 or scFvMut 5 (2 μM). Absorption was measured continuously for 2 h at 37°C (left panel). Activation measurement of recombinant caspase-3 by scFv 5 or scFvMut 5 in the presence of Mg²⁺ using Granzyme B as positive and PBS as negative control. After 30 min incubation at 37°C, Glo reagent was added and bioluminescence measured after 30 min incubation at RT (right panel).